The present invention concerns a method for making epothilones and epothilone analogs, and compounds made by the method.
Epothilones A (2) and B (4) were discovered by Hxc3x6fle and coworkers while examining metabolites of the cellulose-degrading myxobacterium Sorangium cellulosum (Myxococcales) as potential antifungal agents. Hxc3x6fle, G.; Bedorf, N.; Gerth, H.; Reichenbach (GBF), DE-B 4138042, 1993 (Chem. Abstr. 1993, 120, 52841). Hxc3x6fle, G.; Bedorf, N.; Steinmeth, H.; Schomburg, D.; Gerth. H.; Reichenbach, H. Angew. Chem. Int. Ed. Engl. 1996, 35, 1567. 
Initial investigations by scientists at the Gesellschaft fxc3xcr Biotechnologische Forschung in Germany concerned the action of epothilones against fungi, bacteria, and a variety of animal cell lines. Hxc3x6fle, G. et al., Chem. Abstr., 1993, 120, 52841. The epothilones tested had only a narrow spectrum of antifungal activity, but had a rather dramatic effect against oomycetes, such as Phytophotora infestans, the causative species of potato-blight disease. Nicolaou, K. C. et al., xe2x80x9cChemical Biology of Epothilones,xe2x80x9d Angew. Chem. Int. Ed., 1998,37, 2015, which is incorporated herein by reference.
Although the antifungal spectrum of 2 and 4 proved to be quite narrow, scientists at Merck found that these macrolides are highly cytotoxic. Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995, 55, 2325. The epothilones had powerful activity against mouse fibroblast and leukemia cells (2 ng mLxe2x88x921) and strong immunosuppressive activity. Gerth, K., et al., Antibiot., 1996, 49, 560-563. By observing the effect of the epothilones on induction of tubulin polymerization to microtubules and noting that 2 and 4 are competitive inhibitors of Taxol with almost identical IC50 values, it was concluded that epothilones act at the cellular level by a mechanism similar to Taxol. Bollag, D. M. Exp. Opin. Invest. Drugs 1997, 6, 867; Nicolaou, et al., Angew. Chem. Int. Ed. Engl., supra. Epothilone B (2) was particularly impressive in these assays, having a 2,000-5,000-fold higher potency than Taxol in multiple-drug-resistant cell lines. Bollag, D. M.; et al., Cancer Res. 1995, supra.
After scientists from Merck reported their findings on the mode of action of epothilones in 1995, interest in these compounds increased. The Merck scientists subjected tens-of-thousands of compounds to biological assays for Taxol-like tubulin-polymerization activity. Their only hits were epothilones A and B.
Tubulin polymerization-depolymerization plays an important role in the cell cycle, particularly during mitosis. Tubulin is a heterodimer protein comprising globular xcex1,xcex2-tubulin subunits. Tubulin is the monomeric building block of microtubules. Microtubules are one of the fundamental structural components of the cytoskeleton in all eukaryotic cells. Microtubules help develop and maintain the shape and structure of the cell as needed. They may operate alone, or in conjunction with other proteins to form more complex structures, such as cilia, centrioles, or flagella. Nicolaou et al., at 2019, supra.
Structurally, microtubules are regular, internetworked linear polymers (protofilaments) of highly dynamic assemblies of heterodimers of xcex1 and xcex2 tubulin. Nicolaou et al., supra. When thirteen of these protofilaments are arranged parallel to a cylindrical axis they self-assemble to form microtubes. These polymers form tubes of approximately 24 nm in diameter and up to several xcexcm in length. Nicolaou et al., supra.
The growth and dissolution of microtubules are regulated by bound GTP molecules. During polymerization, GTP molecules hydrolyze to guanosine diphosphate (GDP) and orthophosphate (Pi). The half-life of tubulin at 37xc2x0 C. is nearly a full day, but that of a given microtubule may be only 10 minutes. Consequently, microtubules are in a constant state of flux to respond to the needs of the cell. Microtubule growth is promoted in a dividing or moving cell, but is more controlled in a stable, polarized cell. The regulatory control is exerted by adding (for growth) or hydrolyzing (for shrinkage) GTP on the ends of the microtubule.
Microtubules are major components of the cellular apparatus and play a crucial role in mitosis, the process during cell replication in which the duplicated genetic material in the form of chromosomes is partitioned equally between two daughter cells. When cells enter mitosis, the cytoskeletal microtubule network (mitotic spindle) is dismantled by melting at the center, and two dipolar, spindle-shaped arrays of microtubules are formed outwards from the centrosome. Nicolaou et al., at 2020, supra. In vertebrate cells, the centrosome is the primary site of microtubule nucleation (microtubule-organizing center or MTOC). At metaphase, the dynamic action of the microtubules assembles the chromosomes into an equatorial position on the mitotic spindle. At anaphase, the microtubule dynamics change and the chromosomes partition and move to the new spindle poles on the dynamic microtubules, where the new cells are being formed. Nicolaou et al., supra. By this process, the parent cell duplicates its chromosomes, which provides each of the two daughter cells with a complete set of genes. When it is time for a eukaryotic cell to divide, microtubules pull its chromosomes apart and pushes them into the two emerging daughter cells. The rate at which microtubules change their length increases by 20- to 100-fold during mitosis relative to the rate during interphase. These rapid dynamics are sensitive to tubulin-interactive agents which exert their antimitotic action at the metaphase-to-anaphase transition. Kirschner et al., Cell, 1986, 45, 329-342.
A number of anticancer drugs having diverse molecular structures are cytotoxic because they disrupt microtubule dynamics. Most of these compounds, including known chemotherapeutic agents colchicine, colcemid, podophyllotoxin, vinblastine, and vincristine, interfere with the formation and growth of microtubules and prevent the polymerization of microtubules by diverting tubulin into other aggregates. This inhibits cell proliferation at mitosis.
Vinblastine binds to the ends of microtubules. Vinblastine""s potent cytotoxicity appears to be due to a relatively small number of end-binding molecules. Mitchison et al., Nature, 1984, 312, 237-242.
Colchicine first binds to free tubulin to form complexes. These complexes are incorporated into the microtubules at the growth ends in relatively low concentrations, but show profound effects on the microtubule dynamics. Toso R. J., Biochemistry, 1993, 32, 1285-1293.
Taxol disturbs the polymerization-depolymerization dynamics of microtubules in vitro, by binding to the polymeric microtubules and stabilizing them against depolymerization. Cell death is the net result. Epothilones appear to act by the same mechanism and bind to the same general regions as Taxol does. Bollag et al., Cancer Res., 1995, 55, 2325-2333. Epothilones displace Taxol from its receptor, but bind in a slightly different manner to microtubules, as suggested by their action against Taxol-resistant tumor cells, which contain mutated tubulin. Each tubulin molecule of the microtubules contains a Taxol binding site. Taxol and epothilone binding markedly reduce the rate of xcex1/xcex2 tubulin dissociation.
Merck scientists compared the effects of the epothilones and Taxol on tubulin and microtubules and reported higher potencies for both epothilones A and B as tubulin polymerization agents (epothilone B greater than epothilone A greater than Taxol). All three compounds compete for the same binding site within their target protein. The epothilones exhibit similar kinetics in their induction of tubulin polymerization, and gave rise to microscopic pictures of stabilized microtubules and damaged cells that were essentially identical to those obtained with Taxol. Epothilones are superior to Taxol as killers of tumor cells, particularly multiple drug resistant (MDR) cell lines, including a number resistant to Taxol. In some of the cytotoxicity experiments, epothilone B demonstrated a 2,000-5,000-fold higher potency than Taxol, as stated above. Moreover, in vivo experiments, carried out recently at Sloan Kettering in New York involving subcutaneous implantations of tumor tissues in mice, proved the superiority of epothilone B.
On treatment with epothilones B, cells appear to be in disarray with their nuclei fragmented in irregular shapes and the tubulin aggregated in distinct wedge-shaped bundles. By interacting with tubulin, the epothilones block nuclear division and kill the cell by initiating apoptosis.
Recently, Hamel and co-workers examined the actions of epothilones A and B with additional colon and ovarian carcinoma cell lines and compared them with the action of Taxol. Kowalski R. J., et al., J. Biol. Chem., 1997, 272, 2534-2541. Pgp-overexpressing MDR colon carcinoma lines SW620 and Taxol-resistant ovarian tumor cell line KBV-1 retained susceptibility to the epothilones. With Potorous tridactylis kidney epithelial (PtK2) cells, examined by indirect immunoflourescence, epothilone B proved to be the most active, inducing extensive formation of microtubule bundles. Nicolaou et al., at 2022, supra.
Epothilone A initiates apoptosis in neuroblastoma cells just as Taxol does. Unlike Taxol, epothilone A is active against a Pgp-expressing MDR neuroblastoma cell line (SK-N-SH). And, the efficacy of epothilone was not diminished despite the increase of the Pgp level during administration of the drug.
Taxol molecules bind to microtubules, making cell division impossible, which kills the cells as they begin to divide. Since cancer cells divide more frequently than healthy cells, Taxol damages tumors where runaway cell division occurs most profoundly. Other rapidly dividing cells, such as white blood cells and hair cells, also can be attacked. Consequently, side effects are experienced by patients taking the drug. Chemotherapy with Taxol frequently is accompanied by immune system suppression, deadening of sensory nerves, nausea, and hair loss (neutropenia, peripheral neuropathy, and alopecia).
Taxol exhibits endotoxin-like properties by activating macrophages, which in turn synthesize proinflammatory cytokines and nitric oxide. Epothilone B, despite its similarities to Taxol in its effects on microtubules, lacked any IFN-xcex3-treated murine-macrophage stimulatory activity as measured by nitric oxide release, nor did it inhibit nitric oxide production. Epothilone-mediated microtubule stabilization does not trigger endotoxin-signaling pathways, which may translate in clinical advantages for the epothilones over Taxol in terms of side effects.
The importance of the epothilones as therapeutic agents recently was discussed on the front page of the Jan. 27, 2,000 edition of the Wall Street Journal. This article states:
But Taxol has its drawbacks. Some fast-dividing cancer cells can mutate into forms resistant to the drug. Often, patients with advanced cancer who respond at first to Taxol don""t respond after several cycles of treatment because their cells become resistant, too. Despite conducting dozens of trials over the years, Bristol-Myers has been frustrated in its efforts to expand Taxol""s effectiveness beyond certain breast, ovarian and lung cancers.
That""s why the new drugs, broadly classified as part of a family of chemicals known as the epothilones, hold such promise. In studies not yet published, Bristol-Myers and others have shown that the epothilones disrupt cell division through the same biochemical pathway as Taxol. But for reasons scientists are only beginning to understand, the new drugs are equally effective against cancer cells already resistant to Taxol, as well as cells that develop resistance over time.
Based on the biological activity of the epothilones and their potential as antineoplastics, it will be apparent that there is a need for an efficient method for making epothilones and epothilone analogs. Four total syntheses of 4, and several incomplete approaches, are known. See, for example: (1) Nicolaou, K. C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M. R. V.; Yang, Z. J. Am. Chem. Soc. 1997, 119, 7974; (2) Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kamenecka, T.; Sorensen, E. J.; Danishefsky, S. J. J. Am. Chem. Soc. 1997, 119, 10073; (3) May, S. A.; Grieco, P. Chem. Commun. 1998, 1597; (4) Schinzer, D.; Bauer, A.; Schieber, J. Synlett 1998, 861; (5) Mulzer, J.; Mantoulidis, A. Tetrahedron Lett. 1996, 37, 9179; (6) Claus, E.; Pahl, A.; Jones, P. G.; Meyer, H. M.; Kalesse, M. Tetrahedron Lett. 1997, 38, 1359; (7) Gabriel, T.; Wessjohann, L. Tetrahedron Lett. 1997, 38, 1363; (8) Taylor, R. E.; Haley, J. D. Tetrahedron Lett. 1997, 38, 2061; (9) Brabander, J. D.; Rosset, S.; Bernardinelli, G. Synlett 1997, 824; (10) Chakraborty, J. K.; Dutta, S. Tetrahedron Lett. 1998, 39, 101; (11) Liu, Z.-Y.; Yu, C.-Z.; Yang, J. D. Synlett 1997, 1383; (12) Liu, Z.-Y.; Yu, C.-Z; Wang, R.-F.; Li, G. Tetrahedron Lett. 1998, 39, 5261; (13) Mulzer, J.; Mantoulidis, A.; xc3x96hler, E. Tetrahedron Lett. 1997, 38, 7725; and (13) Bijoy, P.; Avery, M. A. Tetrahedron Lett. 1998, 39 1209.
Methods for making epothilone and epothilone analogs also have been described in the patent literature, including: (1) Schinzer et al., WO 98/08849, entitled xe2x80x9cMethod for Producing Epothilones, and Intermediate Products Obtained During the Production Processxe2x80x9d; and (2) Reichanbach et al., WO 98/22461, entitled xe2x80x9cEpothilone C, D, E, and F, Production Process, and Their Use as Cytostatic as well as Phytosanitary Agents.xe2x80x9d One disadvantage associated with these prior processes for synthesizing epothilones is the lack of stereoselectivity in the production of the Z trisubstituted bond of the desepoxyepothilone. As a result, a new synthetic approach to epothilones and epothilone analogs is required which addresses this and other problems associated with syntheses of the epothilones known prior to the present invention.
The present invention provides a novel method for making epothilones and epothilone analogs. The method can provide almost complete stereoselectivity with respect to producing the Z trisubstituted double bond of the desepoxyepothilone, and therefore addresses one of the disadvantages associated with methods known prior to the present invention.
One embodiment of the method comprises first providing a compound having Formula 1. 
With reference to Formula, 1 R is H or a protecting group; R1 is an aryl group, such as, without limitation, benzene derivatives or the thiazole of epothilone B; R2-R5 substituents independently are selected from the group consisting of H and lower alkyl groups; and R6 substituents independently are selected from the group consisting of lower alkyl groups. Compounds having Formula 1 are then converted into an epothilone or an epothilone analog. For example, in the synthesis of epothilone B the step of converting the compound can involve first removing the protecting groups, and thereafter forming an epoxide at C-12, C-13.
In preferred embodiments, R1 is the thiazole shown below. 
Most known epothilones have this thiazole as the aryl group.
Providing compounds having Formula 1 can be accomplished in a number of ways. One embodiment comprises coupling a first compound having Formula 2, 
where R is H or a protecting group and X is a functional group or chemical moiety equivalent to a carbanion at a terminal carbon of the first compound, with a second compound having Formula 3
With reference to Formula 3, R2 is H or lower alkyl, R3 is H or a protecting group, and Y is an electrophillic group capable of reacting with and coupling to the terminal carbon of the first compound. The precursor compound is then converted into compounds having Formula 1. For example, two compounds, one having Formula 2 and the other Formula 3, can be coupled by a Wittig reaction where X is PPH3+ and Y is a carbonyl compound, such as an aldehyde.
Compounds having Formula 1 can be provided by a second embodiment of the present invention. This second embodiment involves coupling a first compound having Formula 4
where R is H or a protecting group and X is a halide, with a second alkyne compound having Formula 5
where R1 is H or a protecting group and R2 is H or lower alkyl. This compound is then converted into a compound having Formula 1. This second embodiment can proceed by first forming an enyne precursor compound having Formula 6
where the substituents are as stated above.
Still another embodiment of the method of the present invention for forming epothilones or epothilone analogs comprises forming the precursor enyne compound having Formula 6 where R1 is H or a protecting group, or a triene compound having Formula 7
where R1 is H or a protecting group, R2 is H or lower alkyl, and R3 is H or a protecting group. Compounds having Formulas 6 and/or 7 are then converted into a compound having Formula 8, where the carbon atom numbers correspond to the numbering system stated for epothilone A. 
With reference to Formula 8, R-R7 are independently selected from the group consisting of H, lower aliphatic groups, particularly lower alkyl groups, protecting groups, or are bonded to an O in an epoxide or an aziridine. More particularly, R substituents independently are H, lower alkyl, or a protecting group; R1 is an aryl group; R2 is H or lower alkyl; C13 and C12 are carbons bonded together by a single bond or a double bond; R3 and R4 independently are H, lower aliphatic groups, or are bonded to O in an epoxide or to N in an aziridine; C10 and C9 are carbons in a double bond or triple bond, and, where C10 and C9 are carbons in a double bond, R5 and R6 independently are H, or lower aliphatic; and R7 substituents independently are selected from the group consisting of lower aliphatic groups. The configuration of the double bond between C10 and C9 may be cis or trans or E or Z. Compounds having Formula 8 are then converted into an epothilone or an epothilone analog. Moreover, the compound having Formula 6 may be converted into the compound having Formula 7, such as by catalytic semi-hydrogenation. Lindlar""s catalyst has proven an effective catalyst for conducting this catalytic semi-hydrogenation.
The method of the present invention differs from other pathways by assembling the macrolide from two segments, which first are connected at C-9, C-10 before macrolactonization. With reference to the first embodiment of the present invention, fragments were constructed around a preformed Z trisubstituted alkene to circumvent stereochemical problems afflicting known synthetic methods. The 9,10 olefin produced by coupling the two segments confers rigidity on the one portion of the epothilone macrocycle that exhibits flexibility, and hence may be expected to impact its tubulin binding properties. Moreover, this alkene provides a chemical moiety from which novel epothilone analogues can be prepared.
Epothilones, such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, can be made by the method of the present invention. The present invention also provides novel compounds that can be made by the method. These compounds typically have Formula 8
where the substituents are as described above. Preferred compounds satisfying Formula 8 include one or more of the following: (1) R being hydrogen; (2) R1 being the aryl thiazole side chain of the epothilones; (3) R2 being hydrogen or methyl; (4) R3-R6 being hydrogen or methyl, or R3 and R4 and/or R5 and R6 being bonded to oxygen in an epoxide; (5) R7 being methyl.
Compounds having Formula 8 include several chiral centers, which allows for a plurality of diastereomers. The present invention is directed to all such stereoisomers. But, the epothilones have known stereochemistries at each of the chiral centers. As a result, preferred compounds of the present invention have the same stereochemistries at each chiral center as do the epothilones. This is illustrated below in Formula 9, which shows the stereochemistries of preferred epothilone analogs at each chiral center. 
The process of the present invention can be used to make known epothilones A, B, C, D, E and F, as well as analogs of these compounds, including the cryptothilones, which typically are dilactone or lactone-amide-type analogs of the epothilones. The cryptothilones are hybrid structures which include a portion of cryptophycins and a portion of the epothilones. One such novel diene analog 10 has double bonds at positions C-9, C-10, and C-12, C-13, including all combinations of cis (10) and trans (11) (Z and E) double bonds 
Using compound 10 and/or 11 to make analogs of epothilones, such as the cryptothilones, provides advantages relative to prior known syntheses, as indicated above.
A method for making diene 10 and converting 10 into, for example, epothilone B, as well as other epothilones and epothilone analogs, is described below.
Formula 8 is a generic structural formula for diene and enyne derivatives of Compound 10. 
Preferred compounds have the stereochemistries shown in Formula 9. 
With reference to Formulas 8 and 8, R is H, lower aliphatic, preferably lower alkyl, or a protecting group; R1 is an aryl group; C13 and C12 are carbons bonded together by a single or double bond; R3 and R4 independently are H, lower alkyl, or are bonded to oxygen in an epoxide or to nitrogen in an aziridine; C10 and C9 are carbons in a single bond, double bond or triple bond, with preferred compounds having C10 and C9 bonded together by a double bond or a triple bond; if C10 and C9 are bonded together by a double bond, the configuration of the double bond may be cis or trans or E or Z; and R5 and R6 independently are H, lower aliphatic, preferably lower alkyl, or are bonded to heteroatoms in cyclic structures, such as to oxygen in an epoxide or to nitrogen in an aziridine.
As used herein, xe2x80x9clowerxe2x80x9d refers to carbon chains having 10 or fewer carbon atoms, typically less than 5 carbon atoms. xe2x80x9cLower aliphaticxe2x80x9d includes carbon chains having: (a) sites of unsaturation, e.g., alkenyl and alkynyl structures; (b) non-carbon atoms, particularly heteroatoms, such as oxygen and nitrogen; and (c) all branched-chain derivatives and stereoisomers.
The phrase xe2x80x9cprotecting groupxe2x80x9d is known to those of ordinary skill in the art of chemical synthesis. xe2x80x9cProtecting groupxe2x80x9d refers generally to a chemical compound that easily and efficiently couples to a functional group, and can be easily and efficiently removed to regenerate the original functional group. By coupling a protecting group to a first functional group of a compound other functional groups can undergo chemical or stereochemical transformation without affecting the chemistry and/or stereochemistry of the first functional group. Many protecting groups are known and most are designed to be coupled to only one or a limited number of functional groups, or are used for particular circumstances, such as reaction conditions. Theodora Greene""s Protecting Groups in Organic Syntheses, (Wilely Science, 1984), and later editions, all of which are incorporated herein by reference, discuss protecting groups commonly used in organic syntheses. Examples of protecting groups used to protect hydroxyl functional groups for the syntheses of epothilones and epothilone analogs include the silyl ethers, such as t-butyl dimethyl silyl (TBDMS) ethers, and tetrahydropyranyl (THP) ethers.
xe2x80x9cArylxe2x80x9d refers to compounds derived from compounds having aromatic properties, such as benzene. xe2x80x9cArylxe2x80x9d as used herein also includes compounds derived from heteroaromatic compounds, such as oxazoles, imidazoles, and thiazoles.
Preferred aryl groups have Formula 10
where X and Y are independently selected from the group consisting of heteroatoms, particularly oxygen, nitrogen and sulfur. For the epothilones, and most epothilone analogs, the R1 aryl group is thiazole 18 shown below. 
C13 and C12 of Formula 8 are carbons bonded together by a single or double bond. Whether C13 and C12 are joined by a single or double bond determines, in part, substituents R3 and R4. For example, if C13 and C12 are coupled by a single bond, then R3 and R4 are selected from the group consisting of hydrogen and lower alkyl. Moreover, if C13 and C12 are coupled by a single bond then R3 and R4 can be bonded to a heteroatom, such as oxygen and nitrogen, in a cyclic structure, such as an epoxide or an aziridine. Epoxide 20 and aziridine 22 are examples of these compounds. 
Wavy and straight bonds to carbons at chiral centers of these structures indicate that all stereoisomers are within the scope of the present invention. With respect to the aziridine analogs, such as aziridine 22, R2 is selected from the group consisting of hydrogen, lower aliphatic, particularly lower alkyl, acyl, and aryl. Preferred compounds have R2 be hydrogen or lower alkyl.
C10 and C9 of Formula 8 are carbons bonded together by a single, double or triple bond. Whether C10and C9 are joined by a single bond, a double bond or a triple bond determines, in part, substituents R5 and R6. For example, if C10 and C9 are coupled by a single bond, then R5 and R6 typically are selected from the group consisting of hydrogen and lower aliphatic, preferably lower alkyl. Moreover, if C10 and C9 are coupled by a single bond then R5 and R6 also can be bonded to a heteroatom, such as oxygen and nitrogen, in a cyclic structure, such as an epoxide or an aziridine. Epoxide 24 and aziridine 26 provide examples of these compounds. 
Compounds 28, 30, 32 and 34 provide additional examples of epoxide/aziridine epothilone analogs. 
Known epothilones have significant biological activity. Novel epothilone analogs made according to the method of the present invention also have been shown to have significant biological activity. For example, Table I provides biological data for certain epothilones and epothilone analogs.
The antiproliferative activity of cis 9,10-dehydroepothilone D and trans 9,10-dehydroepothilone D was assessed in vitro using a panel of human cancer cell lines. As illustrated in Table 1, cis 9,10-dehydroepothilone was 20- to 30-fold less potent than natural epothilone D, and 330-to 670-fold less potent than epothilone B. Interestingly, trans 9,10-dehydroepothilone D showed biological activity very similar to that of its cis isomer in spite of an apparent difference in the conformation of these two macrolactones. Thus, the average IC50 of trans 9,10-dehydroepothilone D for growth inhibition in the cell line panel used in this study was only 1.36-fold higher than that observed for cis 9,10-dehydroepothilone D. As noted for epothilones B and D, cis 9,10-dehydroepothilone D and trans 9,10-dehydroepothilone D retain full anti-proliferative activity against KB-8511 cells, a paclitaxel-resistant cell line overexpressing P-glycoprotein (Table 1). While the tubulin polymerization activity of cis 9,10-dehydroepothilone D and trans 9,10-dehydroepothilone D was lower than of natural epothilone D (56%, 36%, and 88%, respectively) (Table 1), it is conceivable that decreased cellular penetration may contribute to the reduction in antiproliferative potency observed for cis 9,10-dehydroepothilone D and trans 9,10-dehydroepothilone D. The absence of a clear difference in the biological profiles of cis and trans analogues of 9,10-dehydroepothilone D observed here has a parallel in results previously reported for other epothilone analogs. Thus, epothilones incorporating a trans epoxide or trans olefin at C12-C13 have been shown to possess biological activity comparable to their cis isomer.
Taken together, these data support the proposition that the C8-C13 region of the epothilone perimeter is relatively tolerant of structural modification and suggest that the interaction of this segment of the molecule with tubulin is less stringently defined.
The synthesis of epothilones can be exemplified by a working embodiment of a method for making epothilone B. Epothilone B was synthesized by coupling a first subunit with a second subunit to form a coupled intermediate for forming epothilones. One embodiment of the method comprised coupling a first subunit 36 with a second subunit 38. 
A second embodiment comprised coupling a first allylic halide subunit 40 with a second alkyne subunit 42
With respect to 36, 38, 40, and 42, the R substituents are as described above.
A first embodiment of a the present method for making epothilones and epothilone analogs comprised making a suitable subunit 36 as illustrated by Scheme 1, i.e., compound 60. 
Synthesis of segment 36 began from (Z)3-iodo-2-methyl-2-propen-1-ol prepared in geometrically pure form from propargyl alcohol. After protection to provide 44, the iodoalkene was converted to the corresponding cuprate, which underwent clean conjugate addition to (S)-3-acryloyl-4-benzyl-2-oxazolidinone (45) to yield 46. Hydroxylation of the sodium enolate derived from 46 with Davis oxaziridine gave 48. (See, for example, Evans et. al., Chem. Int. Ed Engl., Vol. 26, p. 2117, 1997). The configuration of 48 was confirmed by oxidative degradation to dimethyl (S)-malate. Protection of alcohol 48 as silyl ether 50, followed by exposure to catalytic potassium thioethoxide in ethanethiol provided 52, along with recovered oxazolidinone (93%). Treatment of thioester 52 with lithium dimethylcuprate furnished ketone 54, which upon Horner-Emmons condensation with phosphonate 53 (shown below) produced diene 56 in excellent yield, accompanied by 5% of its (Z,Z) isomer. The tetrahydropyranyl ether protecting group was removed using magnesium bromide. The liberated alcohol was converted to bromide 58. Homologation of 58 to phosphonium bromide 60 using triphenylmethylenephosphorane completed the synthesis of segment 36.
One embodiment of a segment 38, i.e., compound 74, was made as shown by Scheme 2. 
A key construction in one embodiment of a suitable segment 36 involved an aldol condensation of ketone 62 with aldehyde 64. This double stereodifferentiating reaction proceeded in good yield to give anti-Felkin product 66 as the sole stereoisomer. An important contribution to the stereoselectivity of this condensation is made by the p-methoxybenzyl (PMB) ether of 64, since the TBS protected version of this aldehyde resulted only in a 3:2 mixture of 66 and its Felkin diastereomer, respectively. The favorable outcome with 64 is consistent with chelation of the aldehyde carboxyl with both the lithium enolate from 62 and the PMB ether. After protection of 66 as tris ether 68, the terminal olefin was cleaved oxidatively to carboxylic acid 70, which was converted to its methyl ester 72. Hydrogenolysis of the PMB ether and oxidation of the resultant alcohol 74 yielded aldehyde 76.
Subunits 60 and 76 were coupled together, followed by macrolactonization, to provide the diene lactone precursor to epothilone B as shown below in Scheme 3. 
Wittig coupling of the ylide from 58, compound 60, with aldehyde 76 at low temperature afforded triene 78 as a single stereoisomer in excellent yield. Selective removal of the C-15 silyl ether of 78 was unsuccessful. But, after saponification to carboxylic acid 80 this deprotection was readily accomplished with tetra-n-butylammonium fluoride. Macrolactonization of seco acid 82 was carried out under Yamaguchi""s conditions and both silyl ethers were cleaved with acid to yield 9,10-dehydrodes-epoxyepothilone B 84.
Compounds made in this manner can be converted to epothilones using conventional chemistry. For example selective hydrogenation of the disubstituted olefin of 84 with diimide gave the known lactone 86. Lactone 86 underwent epoxidation with dimethyidioxirane to produce 4. Epoxidation can be accomplished according to the method of Danishefsky et al., Angew. Chem., 1997, 109, 775; and Angew. Chem. Int. Ed. Engl., 1997, 36, 757, both of which are incorporated herein by reference. Characterization data for both 86 and 4 matched those in the literature and/or of the naturally occurring product. The 1H NMR spectrum of 4 was in excellent agreement with that provided by Professor Grieco.
Schemes 1-3 provide a convergent synthesis of epothilone B (2), which generates all seven of its asymmetric centers in a completely stereoselective fashion. In addition, clean Z configuration at the C-12, C-13 double bond is incorporated by this pathway. Finally, the Z olefin at C-9, C-10 provides a chemical moiety from which exploratory structural modifications can be made.
Scheme 4 illustrates a second embodiment of a method for making epothilones and epothilone analogs. 
With reference to Scheme 4, compound 76 was made as illustrated above in Scheme 2, and as discussed in more detail in Example 16. Aldehyde 76 was reacted with dimethyl diazophosphonate [J. C. Gilbert et al., J. Org. Chem., 1982, 47, 1837] in THF at xe2x88x9278xc2x0 C. to provide alkyne 88 in approximately 80% yield. The copper (I) derivative of alkyne 88 was produced and was found to couple with allylic halide 58. This reaction was extensively investigated, and was found to proceed to product 90 best when the conditions for the reaction were as shown in Table 2, using about 5% CuI, Et3N, Et2O-DMF, and about 2.0 equivalents of 88. Conditions investigated for this coupling are summarized below in Table 4.
Product 90 was semi-hydrogenated over Lindlar""s catalyst [Pd/CaCO3, Pd(OAc)2]. This reaction was found to proceed best when hexanes was used as the solvent. The hydrogenated product was then saponified using NaOH and isopropyl alcohol at 45xc2x0 C. to provide the corresponding seco acid 80 in approximately 66% yield. The C-15 TBS ether 80 was then deprotected using TBAF and THF by warming the reaction from 0xc2x0 C. to 25xc2x0 C., with a yield of about 89%. The selectivity of this reaction is attributed to sterically favorable transilyation involving the carboxylate anion. The resultant silyl ester is hydrolyzed during aqueous work-up. Macrolactonization was then performed under Yamaguchi conditions. Yamaguchi et al., Bull Chem. Soc. Jpn, 1970, 52, 1989. The remaining TBS ether protecting groups were then removed using trifluoroacetic acid (TFA) in dichloromethane at 0xc2x0 C. to provide compound 84. Compound 84 was then converted to 4 as discussed with respect to Scheme 3 and Examples 21, 22 and 23.
Schemes 5, 6, and 7 illustrate an embodiment of a synthesis via Stille coupling that yields epothilone derivatives containing a trans (or E) double bond between C9 and C10 (See Formula 8).
With reference to Scheme 5, compound 92 was esterified with 2-(trimethylsilyl)ethanol using Mitsunobu conditions to provide 94. Hydrogenolysis removed the p-methoxybenzyl ether from 94, and oxidation of alcohol 96 afforded an aldehyde which was reacted with Bestmann""s reagent (Mxc3xcller et al., Synlett, p. 521, 1996) to give terminal alkyne 96. Hydrostannylation of the latter in the presence of a palladium dichloride catalyst furnished vinylstannane 98.
With reference to Scheme 6, compound 100, which was protected as TES ether 102 with triethylsilyl triflate. The latter was advanced to alcohol 104 by a four-step sequence analogous to that used for the conversion of 48 to 56 (see Scheme 1) and including a final step of removing the tetrahydropyranyl ether protecting group with magnesium bromide. For Stille coupling purposes, the allylic chloride 106 derived from 104 was found to be more effective than the corresponding bromide (Scheme 1).
Coupling of 98 with 106 (Scheme 7) in the presence of catalytic dipalladium tris(dibenzylideneacetone)chloroform complex and triphenylarsine (Farina. and Krishnan, J. Am. Chem. Soc., 113: 9585, 1991) proceeded in high yield and gave the 9E, 12Z, 16E-heptadecanoate 108. Exposure of 108 to tetra-n-butylammonium fluoride cleaved both the (trimethylsilyl)ethyl ester and the triethylsilyl ether but left tert-butyidimethylsilyl ethers at C3 and C7 intact. The resulting seco acid 110 underwent facile macrolactonization to 112, and subsequent removal of the remaining pair of TBS ethers with trifluoroacetic acid furnished trans 9,10-dehydroepothilone D (114). 